Effect of solution properties on the counting and sizing of subvisible particle standards as measured by light obscuration and digital imaging methods.

PURPOSE

Protein formulations may contain subvisible particle (SbVP) impurities that can vary (e.g., in number, size, shape, density, refractive index and transparency) depending on the formulation composition, environmental stresses and the type of protein. Additionally formulation solutions may differ in their physical properties including turbidity, color, viscosity, density and refractive index. This study examined the impact of these formulation matrix parameters on the ability to size and count subvisible particles using a variety of analytical methods including two light obscuration (HIAC, Syringe) and two digital imaging instruments (MFI®, FlowCAM®). Several subvisible particle standards were tested, including polystyrene and glass beads as well as a new pseudo-protein particle standard, in order to also study of the effect of subvisible particles with different properties.

RESULTS

The color and turbidity of solutions generally had a relatively small effect on SbVP sizing and counting. Solution viscosity and refractive index (RI), however, showed a more pronounced effect on the analytical results, especially with more translucent particles such as glass beads and the "pseudo protein standards", resulting in smaller sizes and lower counts of SbVPs, especially when measuring particles using light obscuration methods.

CONCLUSIONS

Each instrument showed certain advantages and disadvantages depending on the analytical parameter (i.e., accuracy, precision), type of subvisible particle, and solution properties. Based on these results, it is recommended to not only carefully consider physical solution parameters as part of analytical method assessment for counting and sizing SbVP in protein dosage forms, but also in terms of various typical QC validation parameters using actual protein formulations.